Engineering Halomonas bluephagenesis for Pilot Production of Terpolymers Containing 3-Hydroxybutyrate, 4-Hydroxybutyrate and 3-Hydroxyvalerate) from Glucose.

Abstract

Microbial poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate), abbreviated as P(3HB-4HB-3HV) or P34HBHV, is a flexible polyhydroxyalkanoate (PHA) material ranging from softness to elasticity depending on the ratios of various monomers. Halomonas bluephagenesis, as the chassis of the next generation industrial biotechnology (NGIB) able to grow contamination free under open unsterile conditions. The resulting recombinants of H. bluephagenesis became capable of efficiently synthesizing P34HBHV utilizing glucose as the sole carbon source. Engineered H. bluephagenesis H1 (encoding ogdA, sucD, 4hbD, orfZ, scpA and scpB in chromosomes) transformed with a plasmid containing PHA synthesis genes phaC and phaA and its derivative H29 produced up to 92% P(3HB-co-8.85%4HB-co-8.47%3HV) and 72% P(3HB-co-13.21%4HB-co-11.97%3HV) in cell dry weight (CDW), respectively, in shake flasks. In bioreactor cultivation, H. bluephagenesis H39 constructed by integrating the 4hbD, phaC and phaA genes into the genome of H. bluephagenesis H1 achieved 95 g/L CDW with 69% P(3HB-co-10.49%4HB-co-3.54%3HV), while H. bluephagenesis H43, further optimized with lpxM deletion, reached 73 g/L CDW with 78% P(3HB-co-10.35%4HB-co-4.54%3HV) in a 100 L bioreactor. For the first time, H. bluephagenesis was successfully engineered to generate stable and hyperproductive derivative strains for pilot production of P(3HB-4HB-3HV) with customizable monomer ratios from glucose as the sole carbon source.